scholarly journals SARS-CoV-2 ORF8 encoded protein contains a histone mimic, disrupts chromatin regulation, and enhances replication

2021 ◽  
Author(s):  
John Kee ◽  
Samuel Thudium ◽  
David Renner ◽  
Karl Glastad ◽  
Katherine Palozola ◽  
...  
Keyword(s):  
Host Cell ◽  
Transcriptional Response ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Alveolar Type ◽  
High Morbidity ◽  
Histone Proteins ◽  
Global Pandemic ◽  
Associated Proteins ◽  
Induced Pluripotent

SARS-CoV-2 emerged in China at the end of 2019 and caused the global pandemic of COVID-19, a disease with high morbidity and mortality. While our understanding of this new virus is rapidly increasing, gaps remain in our understanding of how SARS-CoV-2 can effectively suppress host cell antiviral responses. Recent work on other viruses has demonstrated a novel mechanism through which viral proteins can mimic critical regions of human histone proteins. Histone proteins are responsible for governing genome accessibility and their precise regulation is critical for the ability of a cell to control transcription and respond to viral threats. Here, we show that the protein encoded by ORF8 (Orf8) in SARS-CoV-2 functions as a histone mimic of the ARKS motif in histone 3. Orf8 is associated with chromatin, binds to numerous histone-associated proteins, and is itself acetylated within the histone mimic site. Orf8 expression in cells disrupts multiple critical histone post-translational modifications (PTMs) including H3K9ac, H3K9me3, and H3K27me3 and promotes chromatin compaction while Orf8 lacking the histone mimic motif does not. Further, SARS-CoV-2 infection in human cell lines and postmortem patient lung tissue cause these same disruptions to chromatin. However, deletion of the Orf8 gene from SARS-CoV-2 largely blocks its ability to disrupt host-cell chromatin indicating that Orf8 is responsible for these effects. Finally, deletion of the ORF8 gene affects the host-cell transcriptional response to SARS-CoV-2 infection in multiple cell types and decreases the replication of SARS-CoV-2 in human induced pluripotent stem cell-derived lung alveolar type 2 (iAT2) pulmonary cells. These findings demonstrate a novel function for the poorly understood ORF8-encoded protein and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Finally, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.

scholarly journals SARS-CoV-2 protein encoded by ORF8 contains a histone mimic that disrupts chromatin regulation

2021 ◽  
Author(s):  
John Kee ◽  
Samuel Thudium ◽  
Karl Glastad ◽  
Katherine Palozola ◽  
Zhen Zhang ◽  
...  
Keyword(s):  
Host Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Severe Illness ◽  
Global Changes ◽  
Alveolar Type ◽  
High Morbidity ◽  
Histone Proteins ◽  
Post Translational Modifications ◽  
Induced Pluripotent

Abstract SARS-CoV-2 emerged in China at the end of 2019 and caused the global pandemic of COVID-19, a disease with high morbidity and mortality. While our understanding of this novel virus is rapidly increasing, gaps remain in our understanding of how SARS-CoV-2 can effectively suppress host cell antiviral responses. Recent work on other viruses has demonstrated a novel mechanism through which viral proteins can mimic critical regions of human histone proteins. Histone proteins are responsible for governing genome accessibility and their precise regulation is critical for a cell’s ability to control transcription and respond to viral threats. Here, we show that the protein encoded by ORF8 (Orf8) in SARS-CoV-2 functions as a histone mimic of two critical histone 3 sites containing an ARKS motif. Orf8 expression in cells disrupts multiple critical histone post-translational modifications (PTMs) while Orf8 lacking this histone mimic motif does not. Orf8 binds to numerous histone-associated proteins and to DNA, and is itself acetylated within the histone mimic site. Importantly, SARS-CoV-2 infection of multiple susceptible cell types causes the same global changes of histone post-translational modifications that are disrupted by Orf8 expression; these include induced pluripotent stem cell-derived alveolar type 2 cells (iAT2) and cardiomyocytes (iCM) and postmortem patient lung tissue. These findings demonstrate a novel function for the poorly understood SARS-CoV-2 ORF8 encoded protein and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Notably, this work provides a potential mechanism for emerging findings from human patients indicating that ORF8 deletion results in less severe illness and describes a potentially druggable pathway that may contribute to the virulence of SARS-CoV-2.

scholarly journals SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes

2021 ◽  
Vol 118 (16) ◽  
pp. e2022643118
Author(s):  
Yize Li ◽  
David M. Renner ◽  
Courtney E. Comar ◽  
Jillian N. Whelan ◽  
Hanako M. Reyes ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Innate Immune ◽  
Alveolar Type ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Protein Kinase R ◽  
Double Stranded Rna ◽  
Induced Pluripotent ◽  
Host Virus Interactions

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase–ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2–infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host–virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

scholarly journals Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

2020 ◽  
Author(s):  
Elliot W. Swartz ◽  
Greg Shintani ◽  
Jijun Wan ◽  
Joseph S. Maffei ◽  
Sarah H. Wang ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Calcium Flux ◽  
Electrode Arrays ◽  
Spinal Motor Neurons ◽  
Culture Model ◽  
Skeletal Myotubes ◽  
Induced Pluripotent

SummaryThe failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72-associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.

scholarly journals CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

2021 ◽  
Author(s):  
Zhengyu Ouyang ◽  
Nathanael Bourgeois ◽  
Eugenia Lyashenko ◽  
Paige Cundiff ◽  
Patrick F Cullen ◽  
...  
Keyword(s):  
Single Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Model Systems ◽  
Rna Seq ◽  
Cell Type ◽  
Fine Grained ◽  
Single Nucleus ◽  
Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

scholarly journals Present and future challenges of induced pluripotent stem cells

2015 ◽  
Vol 370 (1680) ◽  
pp. 20140367 ◽  
Author(s):  
Mari Ohnuki ◽  
Kazutoshi Takahashi
Keyword(s):  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Disease Modelling ◽  
Future Perspectives ◽  
Differentiated Cells ◽  
Human Ipscs ◽  
Future Challenges ◽  
Induced Pluripotent

Growing old is our destiny. However, the mature differentiated cells making up our body can be rejuvenated to an embryo-like fate called pluripotency which is an ability to differentiate into all cell types by enforced expression of defined transcription factors. The discovery of this induced pluripotent stem cell (iPSC) technology has opened up unprecedented opportunities in regenerative medicine, disease modelling and drug discovery. In this review, we introduce the applications and future perspectives of human iPSCs and we also show how iPSC technology has evolved along the way.

scholarly journals An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

2020 ◽  
Author(s):  
Jerome Robert ◽  
Nicholas Weilinger ◽  
Li-Ping Zao ◽  
Stefano Cataldi ◽  
Emily Button ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Cortical Neurons ◽  
Neurovascular Unit ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Experimental Models ◽  
Flow Conditions ◽  
Anatomy And Physiology ◽  
Induced Pluripotent

Abstract Introduction: The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently noin vitro3-dimensional (3D) perfusible model of the human cortical arterial NVU. Method: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. Results: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It also reproduces key characteristics of cortical neurons and astrocytes, as well as the formation of a selective and functional endothelial barrier. We further provide proof-of-principle that our in vitro human arterial NVU may be suitable to study neurodegenerative diseases such as Alzheimer’s disease (AD), as we report both phosphorylated tau and beta-amyloid accumulation in our model over time. Finally, we show that our arterial NVU model enables the study of neuronal and glial fluid biomarkers. Conclusion: This model is a suitable tool to investigate arterial NVU functions such as neuronal electrophysiology in health and disease. Further the design of platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

scholarly journals Towards an autologous iPSC-derived patient-on-a-chip

2018 ◽  
Author(s):  
Anja Patricia Ramme ◽  
Leopold Koenig ◽  
Tobias Hasenberg ◽  
Christine Schwenk ◽  
Corinna Magauer ◽  
...  
Keyword(s):  
Drug Testing ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Organ Differentiation ◽  
Stem Cell Source ◽  
Organ Systems ◽  
On Chip ◽  
Induced Pluripotent ◽  
Organ Models ◽  
Model Platform

AbstractMicrophysiological systems are fundamental for progressing towards a global paradigm shift in drug development through the generation of patient-on-a-chip models. An increasing number of single- and multi-organ systems have been adopted by the pharmaceutical and cosmetic industries for predictive substance testing. These models run on heterogeneous tissues and cell types from different donors. However, a patient is an individual. Therefore, patient-on-a-chip systems need to be built from tissues from one autologous source. Individual on-chip organ differentiation from a single induced pluripotent stem cell source could provide a solution to this challenge.We designed a four-organ chip based on human physiology. It enables the interconnection of miniaturized human intestine, liver, brain and kidney equivalents. All four organ models were predifferentiated from induced pluripotent stem cells from the same healthy donor and integrated into the microphysiological system. The cross talk led to further differentiation over a 14-day cultivation period under pulsatile blood flow conditions in one common medium deprived of growth factors. This model platform will pave the way for disease induction and subsequent drug testing.

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